1. Field of the Invention
The present invention relates generally to thiopurine drugs used for the treatment of inflammatory bowel disease, leukemia, and organ transplantation rejection, and more specifically to methods for determining thiopurine methyltransferase activity in order to individualize dosages of 6-mercaptopurine therapy.
2. Background Information
Mercaptopurine (6-MP or 6-thiopurine) and azathioprine [6-(1-methyl-4-nitro-5-imidazolylthio)purine] are cytotoxic drugs which are effective in the treatment of ulcerative colitis and Crohn""s disease (Present et al., Annals of Internal Medicine 111:641-649 (1989)). Both are immunosuppressive agents that act as purine antagonists and thereby inhibit the synthesis of DNA, RNA and proteins (Lennard, European Journal of Clinical Pharmacology 43:329-339 (1992)). 6-MP was initially used for the treatment of childhood acute lymphoblastic leukemia and for post-operative treatment of organ transplantation surgery (Burchenal et al., Blood 8:965-999 (1953)), and its use has since been extended to rheumatoid arthritis and inflammatory bowel disease (Kirschner, Gastroenterology 115:813-821 (1998)).
The prodrug azathioprine (AZA) is rapidly converted to 6-mercaptopurine through non-enzymatic, nucleophilic attack by sulfhydryl-containing compounds in the circulation. 6-MP and azathioprine (AZA), which are forms of the same drug and metabolic precursors of the active components, are acted upon by at least three competing enzymatic pathways (Lennard, supra, 1992). An overview of the action of these enzymes is shown in FIG. 1. As shown in FIG. 2, several major enzyme pathways are involved. Xanthine oxidase (XO) converts 6-mercaptopurine to 6-thiouric acid. Hypoxanthine phosphoribosyl transferase (HPRT) converts 6-mercaptopurine to 6-thioinosine-5xe2x80x2-monophosphate, which is a precursor to 6-thioguanine nucleotides. Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of 6-mercaptopurine to methylmercaptopurine (6-MMP). Thus, 6-mercaptopurine is enzymatically converted to various metabolites, including 6-thioguanine (6-TG) and 6-thioguanine nucleotides, which are the presumptive active metabolites mediating the effects of azathioprine/6-mercaptopurine drug therapy.
The interplay of the pathways described above is genetically determined and creates a highly individualized response to azathioprine/6-mercaptopurine drug therapy. The population frequency distribution of TPMT enzyme is trimodal, with the majority of individuals (89%) having high activity, 11% having intermediate activity and about 1 in 300 (0.33%) having undetectable activity (Weinshilboum and Sladek, Amer. J. Human Genetics 32:651-662 (1980)). Such a trimodal relationship has been confirmed by direct measurements of TPMT enzyme activity by the Krxc3x6plin HPLC assay method (Kroplin et al., Eur. J. Clin. Pharmacol., 54 265-271 (1998)). In contrast to variation in TPMT activity, there is very little inter-individual variation in XO activity and only limited data on HPRT activity (Lennard, Eur. J. Clin. Pharm., 43:329-339 (1992)).
Available evidence indicates that TPMT activity effectively modulates the concentration of 6-thioguanine by shunting 6-mercaptopurine into the production of 6-methyl-mercaptopurine. Patients who less efficiently methylate these thiopurines have more extensive conversion to 6-thioguanine nucleotides, which can lead to potentially fatal hematopoietic toxicity. Thus, patients with intermediate or low TPMT activity can be more susceptible to toxic side effects of azathioprine/6-mercaptopurine therapy (Present et al., Annals of Internal Medicine 111:641-649 (1989)). Such toxic side effects include allergic reactions, neoplasia, opportunistic infections, hepatitis, bone marrow suppression, and pancreatitis; in about 1 out of 300 patients, this therapy cannot be tolerated. As a consequence, many physicians are reluctant to treat patients with azathioprine/6-mercaptopurine therapy, particularly due to the risk of infection and neoplasia.
Thus, there is a need for a method of optimizing the dose of 6-mercaptopurine by determining the level of thiopurine methyltransferase activity in a patient. Such a method would be valuable for optimizing therapeutic efficacy of azathioprine/6-mercaptopurine therapy while minimizing undesirable side effects. The present method satisfies this need and provides related advantages as well.
The present invention provides a method of determining thiopurine methyltransferase (TPMT) activity in a subject. The method includes the steps of reacting sample obtained from the subject with a thiopurine derivative that is not 6-mercaptopurine to produce a methylated purine product; contacting the reacted sample with acid, thereby precipitating proteinaceous material from the reacted sample; separating supernatant from the precipitated proteinaceous material; and detecting in the supernatant the methylated purine product, where the amount of the methylated purine product indicates a level of thiopurine methyltransferase activity in the subject. In a method of the invention, the subject can be, for example, an inflammatory bowel disease patient. In one embodiment, the acid used to precipitate proteinaceous material is perchloric acid, for example, 70% perchloric acid. In another embodiment, the thiopurine derivative used as a substrate is 6-thioguanine. In a further embodiment, the methylated purine product is detected by fluorescence, which can be combined, if desired, with high performance liquid chromatography (HPLC).